INNATE IMMUNITY

Innate immune defences consist of:

Recently it has been recognised that phagocytic effector cells of the innate immune system such as macrophages, dendritic cells and NK lymphocytes recognize pathogen associated molecular patterns (PAMPs) via pattern recognition receptors (PRRs) which are germ-line encoded proteins on pathogens. (Table 6.1). The most important of these PRRs are the toll-like receptors (TLRs), although others exist.

Table 6.1 Pattern recognition of receptors of innate immune system

The innate immune system is non-adaptive, i.e. it cannot adapt its receptors to recognise an organism which has evolved and mutated its antigens to evade binding. It does not develop memory (enhanced responses on subsequent encounters with the same antigen), and it does not possess antigen specificity through the specialised and mutable antigen receptors of immunoglobulins, although clearly TLR systems are specific for particular ligands.

SPECIFIC IMMUNE RESPONSES

Specific (adaptive) immune responses are more effective than innate ones and are mediated by lymphocytes and antibodies which amplify and focus non-specific responses and provide additional effector functions. These cells are organised into lymphoid tissues (Fig. 6.2). Humoral immunity often refers to the antibody arm of the specific immune response. Cellular (cell-mediated) immunity refers to lymphocyte-mediated effector responses (T helper (Th) and cytotoxic cells) of the specific immune response. These two arms of the specific immune response are not really separ-able, since antibodies are usually not produced without some cell-mediated response to the same antigen and vice-versa. T and B lymphocytes possess infinitely variable antigen receptors which can clonally expand. Antigen receptors which can be secreted into interstitial fluid and onto mucosal surfaces are called antibodies. Antibodies can activate complement and also enhance opsonisation of antigen to facilitate phagocytosis. Both innate and adaptive mechanisms exponentially amplify the immune response, since clonal expansion of lymphocytes increases the number of cells reactive with an antigen. Cytokines and complement compon-ents recruit other immune effector mechanisms and antibodies activate complement and phagocytes.

Fig. 6.2 The organisation of the lymphoid system. The lymphoid system is organised to ensure efficient recirculation and interaction of T lymphocytes, B lymphocytes and antigen-presenting cells.

IMMUNE RESPONSE

The components of each type of response in the prevention of individual infections or in the pathogenesis of autoimmune diseases differs (Table 6.2).

Table 6.2 Infections and protective immune responses

The specific adaptive immune response is thus flexible and adaptable, capable of responding to antigens which have not been previously encountered, includingthose generated in organisms by the selection pressuresof an effective adaptive immune response. Many pathogens have specific adaptations/mutations to evade previous immunological memory responses (e.g. influenzaantigen variability) or to suppress the normal mechanisms of immune destruction.

COMPLEMENT

The complement system is a soluble enzymic cascade which focuses and amplifies the activity of the specific and innate immune systems as well as having lytic activity against bacteria (Fig. 6.3). It is part of the innate defences since it has no intrinsic antigen specificity.

Fig. 6.3 The complement system. The complement system is an enzymic cascade which leads to the formation of lytic, chemotactic and opsonising factors. ‘Classical’ activation by antibody or ‘alternative’ activation by other means result in similar pro-inflammatory effector function. C = chemotactic activity; O = opsonisation activity; L = lytic activity.

The complement cascade has a final common path-way which leads to the insertion of a multimeric pore-forming structure (membrane associated complex (MAC) consisting of complement components C5-9) into bacterial cell membranes, leading to osmotic lysis. The production of this lytic complex is achieved via two mechanisms called the classical and alternative pathways. Inability to generate the MAC complex leads to particular susceptibility to infections with Neisserial organisms causing recurrent meningitis.

ANTIGENS

An antigen is any substance which can elicit a specific immune response. An antigen consists of many epitopes. An epitope is a specific sequence of a protein or carbohydrate recognised by the receptor molecules of the immune system (antibody or T cell receptor). Antigens can be divided into foreign (non-self, allogeneic, xenogeneic, etc.) and self-antigens (autoantigens). Although an antigen usually elicits an immune response, if the antigen is encountered in appropriate circumstances the specific immune response may be switched off by a variety of mechanisms which will be important to consider when discussing the immunology of transplantation and autoimmune diseases.

ANTIBODIES

An antibody is a soluble protein immune receptor produced by B lymphocytes, consisting of two identical antigen-binding sites (Fig. 6.4). The antigen specificity of the antibody resides in the antigen-binding variable regions (the fragment antigen-binding, Fab, portion). Antibodies are divided into different isotypes (classes) which have different functional attributes due to the Fc (fragment constant) tails coded by the constant region genes of the heavy chain; thus different constant region genes produce different antibody classes. Antibodies which bind to antigen or cells and activate complement via the Fc region thus recruit, activate, amplify and target non-specific defence mechanisms.

Fig. 6.4 The formation of antibodies by somatic recombination. Junctional diversity results from imprecise splicing at the joins between V, D, J and C region genes. This enhances the ability of antibody binding sites (where mutations already occur frequently in hypervariable parts of the V regions) to bind diverse antigens. Heavy chains are formed from V, D, J and C genes, light chains from V, D and C genes. The C region determines the isotype of the heavy chain (G, A, M, E, D) and the light chain (K or L).

Up to 10¹⁰ different antibody specificities may be produced in any individual. This is achieved by joining multiple different copies of genes encoding the variable regions of heavy and light chains of the immunoglobin. Somatic recombination of the gene segments (V, D and J region genes) leads to generation of diversity and broad repertoire of antibody specificities. The antigen-binding variable regions are further (infinitely) diversi-fied by a combination somatic hypermutation (Fig. 6.4) which results from random mutations to the V genes in the hypervariable regions (mutation hotspots) and to the joins between V, D and J genes, enabling antibodies to be produced which can bind to virtually any natural or synthetic antigen encountered. Each cell producing antibody which binds an epitope of an antigen is stimulated to clonally reproduce, and thus further amplification of the immune response occurs with the progeny of each cell producing exactly the same antibody but many different clones expanding.

Most antibody immune responses are polyclonal (many cell clones expand, each recognising different, sometimes overlapping, epitopes on the antigen); oligoclonal responses occur when a limited number of clones expand for some reason (e.g. prolonged inflammation); monoclonal proliferations are usually representative of malignant transformation of a single clone of a B cell at some point in its differentiation (early or late B cells = lymphoma, and often produce IgM; terminally differentiated plasma cells = myeloma and usually produce IgG/A isotypes).

The antigens recognised by antibodies are often conformational (that is, they require a folded 3D structure for recognition), often bringing widely separated areas of a larger molecule together to form the epitope (which is, therefore, discontinuous in linear sequence, unlike the epitopes recognised by T cells). Antibodies thus tend to recognise native folded-3D structures.

Most antibody production is ‘T cell dependent’ (i.e. very inefficient in the absence of T cells, which recognise linear epitopes on the same antigen as that recognised by the antibody and provide ‘help’ (co-stimulation to amplify responses) to B cells,). A small number of relatively ‘T-independent’ B lymphocytes exist which bear the CD5 surface antigen. They tend to recognise conserved carbohydrate epitopes on pathogens (including human ABO blood groups), produce IgM and may represent a phylogenetically older type of B cell defence.

Isotypes and subclasses

B lymphocytes initially produce IgM upon a primary encounter with antigen; this is very efficient at complement fixation and opsonisation, but IgM circulates as a large pentameric (five antibody molecules) structure with a short half-life (∼five days). Subsequently an individual B cell will undergo a class-switch to IgG, IgA, or IgE production, but class-switching depends on effective T cell help following T cell recognition of an epitope on the same antigen. Memory develops in parallel with the class switch. Both these processes require effective communication between B-cells, Antigen Presenting Cells (APC) and T cells (mediated by CD40– CD40L interaction). IgG diffuses well into extracellular spaces and can neutralise circulating viruses and bacteria (prevent binding by blocking receptors), opsonise via complement or Fc receptors or lyse via complement activation. IgG is divided into four subclasses (IgG1, IgG2, IgG3, IgG4) which have different Fc regions (and thus are coded by different heavy chain constant region gene segments). These classes and subclasses have different half lives and abilities to fix complement, or bind Fc receptors (Table 6.3).

Table 6.3 The functional attributes of different immunoglobulin molecules

There are several different types of Fc receptors (FcRI or CD64, FcRII or CD32, FcRIII or CD16) which bind some IgG subclasses better than others and are distributed differently on each effector cell type. IgG1 constitutes 60–70% of the circulating IgG in man; IgG2 constitutes 20–40%. IgG3 constitutes 15–20%. IgG4 circulates in trace amounts and its functional significance is unknown, although it may be important in IgE-mediated antiparasite and allergic responses. IgG1 and IgG3 tend to be produced in response to protein antigens; IgG2 in response to polysaccharide antigens (such as those of bacterial capsules).

IgA is secreted preferentially onto mucosal surfaces and is important in prevention of initial adherence to epithelium or mucosal penetration (blocks interaction with cell surface receptors) of bacterial and viral pathogens spread via respiratory or gastrointestinal routes. IgA deficiency thus predisposes to mucosal infections. The gut contains peptidases which degrade IgG and IgM rapidly. IgA is protected from destruction by a remnant of the polyIg receptor (which selectively transportssecretory IgA across epithelium to the outside of the mucosal surface) called the secretory component, and IgA is usually secreted as a dimer joined by a j(oining)-piece. Most secretory IgA is of the IgA2 subclass; mostcirculating in serum is IgA1. The significance of this is uncertain. Unlike most IgG subclasses or IgM, IgA does not efficiently fix complement via the classical pathway of complement activation.

ANTIGEN-PRESENTING CELLS

In contrast to antibodies, T cells can not recognize native antigens. They recognise short linear peptides on the surface of APC which digest the whole antigen and present the fragments on the surface in the grooves of major histocompatibility complex (MHC) Class I or II molecules (MHC restriction). The initial interaction of T – lymphocytes with antigen is important in determining whether a specific immune response is promoted or suppressed. The default pathway in unprimed ‘naive’ cells (which have not encountered specific antigen before) is either to become specifically unresponsive to the antigen (anergy) or to die (apoptosis) if the antigen is encountered in an insufficiently stimulating context. Naive T lymphocytes are relatively refractory to stimulation, and require potent signals to activate them to clonally proliferate and/or become effector cells. This usually occurs centrally in the lymph nodes, bone marrow or spleen, but can occur elsewhere. These extra signals are complex and multifactorial but act in addition to the recognition of antigenic peptide in the MHC groove by the T cell receptor (TCR) on the CD4 or the CD8 T cell. This incorporates adhesion molecules which stabilise contact between lymphocyte and APC, and costimulator molecules which provide activation signals to the T cell from the APC (Immunological Synapse – cf. neuro-logical synapse). Important interactions occurring at the immunological synapse are shown in Table 6.4.

Table 6.4 Immunological Synapse – crosstalk between APC and T cell

APC of several different types provide these second signals while presenting a processed fragment of antigen to a lymphocyte (Fig. 6.5). Primary stimulation of naive T cells requires a potent professional APC (such as the Dendritic cell (DC) or an activated B lymphocyte) with potent stimulatory capacity and ability to acquire and process (digest) antigen by phagocytosis or endocytosis. Secondary restimulation of recently activated or memory T cells is less stringent and can occur on non-professional APC which are not potent enough to stimulate naive cells effectively, e.g. activated endothelium or monocytes and other cells expressing MHC Class II molecules.

Fig. 6.5 Cellular interactions in adaptive immune responses.

‘Professional’ APC such as DC are resident as sentinels in the skin (Langerhans’ cells) or in the interstitiumof most tissues (interstitial DC) including lymph nodes(interdigitiating DC). On encounter with antigen, DCbecome activated (mature) and migrate centrally via lymphatics to become resident in the T cell areas of lymph nodes (paracortical area) as interdigitating cells. There, T cells recirculating through lymph nodes via lymphatic drainage encounter antigen and clonally proliferate, if they carry the appropriate antigen-specific TCR. Subsequently they migrate back to the peripheral tissues and elicit a local immune response. Similar processes occur in the spleen and Peyer’s patches. B cells may also be stimulated directly by DC.

T LYMPHOCYTES

T cells recognise antigen fragments on the surface of APC which express MHC Class I and II molecules on the surface. MHC molecules have an antigen-binding groove on the surface which can bind antigen fragments of 9–11 amino acids (MHC Class I) or 14–20 amino acids (MHC Class II) in length. Thus they act as display platforms on which the TCR can recognise antigen, but because they bind antigen fragments themselves, the MHC molecules also influence the immune responses in any individual since each MHC type will bind some antigens better than others, and occasionally won’t bind some antigens at all. The TCR binds to a part of the lips of the groove as well as the antigen fragment. Thus the TCR is also self-restricted (MHC Restriction), since it binds only to the combination of [self antigen (MHC) + foreign antigen]. A T cell will not operate effectively with non-self APC which bears different MHC molecules. They can, however, co-operate with non-self cells provided they express the same MHC molecules (as they have to do in allogeneic bone marrow transplantation where the BM is donor-type and the recipient is host-type). MHC Class I is bound by CD8, and MHC Class II by CD4 on the T cell surface (Fig. 6.5). Virtually every nucleated cell expresses MHC Class I on the surface, but MHC Class II expression is restricted to certain cell types (e.g. Professional APC, B lymphocytes) especially when the cell is activated. APC express MHC Class II in high density and thus are the major activators of CD4 positive lymphocytes. MHC Class I restricted CD8 positive T cells are also stimulated by APC, but they recognize foreign peptides (e.g. viral, intracellular bacteria) on all nucleated cells by ‘seeing’ viral antigen in the surface groove of self-MHC Class I, and are activated to deliver a lethal attack on the cell. Not surprisingly, viruses have adapted to reduce MHC Class I surface expression (e.g. adenovirus) and can partially evade their attentions (NB NK cells recognize this lack of MHC class I as a sign of an infected cell). Degraded intracellular antigens in the cytosol tend to get access to the MHC Class I groove in the process of MHC assembly in the endoplasmic reticulum, and thus responses to intracellular antigens tend to occur via the MHC Class I pathway (Fig. 6.6). Extracellular antigens from bacteria phagocytosed and digested in the lysosomes of APC tend to gain access to MHC Class II most (readily since the assembly pathway of MHC Class II molecules intersects with the lysosomal pathway). Thus degraded extracellular antigen gains access to ‘empty’ MHC Class II molecules after the invariant chain (which occupies the MHC groove prior to antigen binding in order to let the molecule pre-assemble without antigen) is displaced by alterations in the intralysosomal pH.

Fig. 6.6 HLA Class I and Class II restricted antigen processing and presentation.

All T cells have CD3 and TCR complex on their sur-face. The T cell receptor requires various co-receptor molecules (LFA-1, CTLA-4, CD28, CD40L) to be associated with it on the cell surface in order to enable efficient antigen recognition and signalling from antigen-presenting cells. Therefore any T cells lacking these co-receptors will fail to function normally.

CD4-bearing T cells generally have ‘helper’ functions;those which aid B cell antibody production are called Th2 and those which activate mononuclear phagocytesand promote cellular inflammatory activity are called Th1 (Table 6.5). These T cells types tend to produce different cytokines when activated by antigen; Th1 produce pro-inflammatory IFNγ, IL-1, IL-12, TNFα; Th2 produce IL-4, IL-5, IL-13, TNFα and others which promote antibody production. In any particular immune response one type of T helper activity will often dominate. This is important both in defence against infection and in the pathogenesis of immunologically-mediated diseases. CD8 positive T cells in contrast often have cytotoxic effector properties and are critically important in defense against certain viral infections.

Table 6.5 T cell subtypes

T CELL STIMULATION – SIGNALS 1 TO 3

The principle of initiation of an immune response is the same whether it is to an infection, to self-antigen in autoimmune diseases or to non-self MHC in an alloimmune response in transplantation. It requires both naïve and memory lymphocytes and the recognistion of antigen through the specific receptor (signal 1) and the simultaneous provision of additionalco-stimulatory signals though other cell surface receptors (signal(s) 2). On internalizing the antigen, APCs become activated and move to the secondary lymphoid organs, bringing the antigen to the central lymphoid system where large numbers of T cells and B cells are present.

The antigen on the surface of dendritic cells triggers the T – cells with an appropriate T – cell receptor which recognizes the MHC-bound antigen fragment and this constitutes ‘Signal 1’, transduced through the TCR-CD3 complex. Co-stimulation or ‘Signal 2’ which is delivered via CD80 (B7.1) and CD86 (B7.2) on APC to CD28 and other molecules on the T-cells. Signals 1 & 2 activate three internal signal transduction pathways:

These pathways lead to activation of transcription factors inducing expression of IL-2, CD-154 and CD25. IL-2 and other cytokines activate the ‘target of rapamycin’ (TOR) pathway to provide ‘Signal 3’, which triggers cell proliferation. This results in the clonal expansion of lymphocytes leading to generation of antibodies and of T-cell effector functions.

Gell and Coombs classified immune responses in the 1930s (Table 6.6). This is now of limited clinical usefulness since mixed patterns are always seen, but is useful for the general understanding of immuno-logically mediated diseases.

Table 6.6 Classification of specific immune (hypersensitivity) responses

SELF-TOLERANCE

T cells recognise antigen together with self-MHC epitopes in the antigen-binding groove of MHC molecules. Strongly self-reactive cells are eliminated (deleted) by encounter with self-antigen on thymic APC (thymic epithelium and DC) in early fetal life. Some self-antigens are probably not expressed in the thymus and remain hidden from the immune system (cryptic epitopes, e.g. intraocular antigens), and tolerance is not established. These antigens tend to reside in immunopriviledged sites, and an immune response does not occur unless released by trauma (e.g. sympathetic ophthalmitis). In adults any cells capable of some degree of self-reactivity which escape deletion in the thymus are probably actively suppressed or made unresponsive (anergised) by peripheral mechanisms which involve T – regulatory cells (CD4+, CD25+ FoxP3 positive T cells). Allograft tolerance can be transferred by these cells (‘infectious tolerance’). The potential for self-reactivity thus exists in all individuals but is usually prevented from becoming a pathogenic mature immune response. Some anti-self immune responses are involved in ‘housekeeping’ activities such as the removal of effete RBC. It is not uncommon to find low titre autoantibodies during infections and tissue injury. Low titres of non-pathogenic autoantibodies (often IgM isotype) are also commonly found in the unwell elderly without any immunological disease.

Autoimmune disease may occur either by reactivation of anergised cells by encounter with potent APCs in certain circumstances which override their programmed unresponsiveness (e.g. where a strong immune response to another antigen results in bystander help sufficient to activate self-reactive T cells in the vicinity), by cross-reactivity between self- and foreign antigens, or as a result of inherited or acquired defects in molecules important in the control of immune responses and maintenance of anergy (e.g. Fas/FasL deficiency-leading to defective apoptosis). The clinical pheno-types of the autoimmunity probably reflect the predominant effector mechanisms and the organ specificity of the antigen(s) and may result in direct damage or interference with normal function. The identity of many autoantigens is now known (Table 6.7). Some are receptors, some enzymes. Autoimmunity may also occur because of failure of induction of self tolerance in the thymus (e.g. autoimmune regulator protein (AIRE) abnormality leading to auto-immune poly-endocrinopathy, candidiasis, ectodermal dysplasia – (APECED)) which results from an inability of the thymic APC to present self antigens to maturing T cells and, therefore, a failure of deletion of self-reactive T cells.

Table 6.7 Autoantigen specificities and disease

Organ-specific autoimmunity manifests itself by damage or malfunction of a single organ as a result of a specific immune response, usually to multiple antigens or to multiple organs on the basis of shared antigens (e.g. steroid cell antibodies linking Addison’s disease and premature ovarian failure, or the lung and kidney damage of Goodpasture’s syndrome). In some conditions (e.g. myasthenia gravis) humoral responses play a major role in many of the disease manifestations, but are unlikely to occur without cellular responses which may also be important. In other diseases, cellular mechanisms may be the predominant pathogenic response: e.g. extrinsic allergic encephalomyelitis (EAE, a model for multiple sclerosis) to myelin basic protein (MBP) and other intracerebral autoantigens. In systemic autoimmunity such as SLE, pathogenesis is multifactorial and involves multiple unrelated antigens. Humoral and cellular responses to multiple nuclear (nucleosome) and cytoplasmic components are seen, particularly anti-double-stranded DNA antibodies (dsDNA) which can cause an immune complex neph-ritis. Sometimes titres of antibodies or complement levels (reduced by immune complex deposition) reflect disease activity in an individual, but in others they do not. In some diseases, the autoantibodies or lymphocytes are pathogenic in models of disease (e.g. anti-dsDNA antibodies in SLE; anti-GBM antibodies in Goodpasture’s). In other diseases they are not, and may be secondary markers of damage (e.g. many antinuclear antibodies (ANA) in SLE, antithyroid peroxidase antibodies in thyroid malignancy).

MHC ANTIGENS AND AUTOIMMUNITY

MHC antigens are inherited (along with a package of minor antigens) as a haplotype consisting of an HLA-A, -B, -C (Class I); -DR, -DP, -DQ (Class II) allele from each parent (Fig. 6.7). Allogeneic immune responses (against a foreign MHC antigen from the same species) can be generated after transplantation.

Fig. 6.7 The structure of the human major histocompatibility gene complex (human leucocyte antigen).

The MHC molecule on the APC determines the type and composition of the peptide fragment that it can present to the naïve T cell, and is an important factor in predisposition, protection or disease expression. Certain alleles or haplotypes are associated with particular diseases (Table 6.8). Both organ-specific and non-organ-specific autoimmune diseases are associated with similar MHC haplotypes in some cases, suggesting an inherited predisposition. Few MHC associations with diseases are very strong (most strongly seen between B27 and ankylosing spondylitis), since most conditions are multifactorial and are a result of a combination of genes and additional envir-onmental influences, perhaps including infection.

Table 6.8 MHC associations with disease

The apparent association of MHC Class I alleles (e.g. HLA-B27) and MHC Class II (e.g. DQB1) may also be due to molecular mimickry between pathogen and MHC, resulting in autoimmune attack. (Heat shock protein (HSP) 60 is widely conserved and generates immune responses in bacterial infection and some autoimmune diseases.). In contrast, some of MHC haplotypes may actually confer protection from some infections and autoimmune disease.

TRANSPLANTATION BARRIER

Non-self antigens are subject to immune-mediated attack by adaptive humoral and cellular mechanisms. The most important antigens are those most widely expressed on the graft, e.g. ABO blood group antigens, and those eliciting strong responses, e.g. disparate MHC antigens (allogeneic response). Any other polymorphic cell surface molecule on the graft which is not expressed by the recipient will also elicit an immune response. In the case of cross-species grafting (xenogeneic transplantation), the rejection response is even stronger as a result of increased disparity between the MHC molecules and the presence of broadly reactive antibodies which bind to the graft and cause hyperacute rejection.

The aim of immunosuppression is to depress the effector immune response to prevent graft rejection (at least initially). The hope is that subsequently either tolerance or graft acceptance will result from downregulation of the antigraft response and enable withdrawal of immunosuppression. The aim of ABO-matching andHLA-matching (tissue typing) is to reduce the antigenic disparity between the graft and the recipient. Other antigens clearly exist (e.g. endothelial antigens) but matching for these is not currently practicable; however, genetic linkage of genes means that related donors with a haplotype match are likely to share the same non-MHC genes. Cyclosporin A (CsA), a fungal metabolite, prolonged survival of renal transplants in man in the late 1970s. By this time, however, graft survival from living related donors had reached a plateau, suggesting that early graft survival results from ABO matching and immunosuppressive drugs, with some contribution from HLA-DR matching (which is more effective than HLA-B or HLA-A matching). Some have, therefore, argued that the benefit of HLA matching is insignificant with modern immunosuppressive drug regimes; however, it appears that long-term graft survival appears more dependent on HLA-A and -B matching (Fig. 6.8).

Fig. 6.8 Renal transplant survival is related to a degree of HLA matching.

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